Co-transporters are an important class of membrane proteins responsible for the accumulation of sugars, amino acids, peptides, neurotransmitters and ions in cells. These proteins are now known to also co-transport water (200-500 molecules per cycle) and small polar molecules such as urea. The energy for the substrate transport comes from the Na+ (or H=) electrochemical potential gradient across the plasma membrane. Our goal is to understand how co-transporters use the free energy released from downhill Na+ transport to drive uphill substrate and water transport. Using the intestinal Na+/glucose co-transporter (SGLT1), and a bacterial homolog (Vibrio SGLT), we plan to identify and determine the architecture of the Na+ and substrate transport pathways, and to determine how Na+ changes the conformation of SGLTs to couple Na+ transport to sugar and water transport. The experimental strategy is to express truncated parts of the co-transporters in Xenopus laevis oocytes and bacteria, and to determine which parts of the protein retain partial reactions, e.g. Na+ uniport, sugar uniport, Na+/sugar co-transport, and water or urea transport. Once parts of the protein are found that exhibit the partial transport reactions, we will use a combination of molecular, biophysical and biochemical techniques to determine their structure and how they interact. Preliminary studies indicate that the C-terminal part of SGLT1 can function as a glucose uniporter, and we have identified several residues in this domain that interact with sugar during Na+/glucose co- transport. The plan is to locate the other residues in the sugar transport pathway, and carry out similar studies on the Na+ transport pathway. These studies will provide a low resolution topological map of the co- transporter and structural information about Na+/sugar/water coupling, and lay the ground work for obtaining higher resolution structures.